We investigated the antiamoebic activity of three Bacteriocin Like Substances (BLS 39, BLS GS 54, BLS GS 16) produced by Lactic Acid Bacteria (LAB). The crude bacteriocins showed an amoebicidal effect against Acanthamoeba polyphaga, but with differences. BLS 39, produced by Lactobacillus pentosus, determined a prompt and progressive decrease of viable amoebal cell count, up to the end of the experiment (144 h), where the trophozoites were not detectable. A killing effect, but after a more prolonged contact time, was observed for BLS GS 54, produced by Lactobacillus paraplantarum, whereas the bacteriocin produced by Lactobacillus plantarum GS16 showed the lowest toxicity for A. polyphaga. For BLS GS 16 the maximum percentage of reduction in trophozoites count (45%) was obtained after 144 h, value much lower when compared to BLS GS54 and BLS 39, that showed values of 44,60% and 52,60% after only one hour of contact, with a maximum of 98% and 100% of non-viable cells, respectively, after 144 h. Morphological changes of the A. polyphaga cells as swollen cells, roundness and cellular lysis, were already observed after the first hours of contact with BLS and, at the end of the experiment, most of the cells were colored (blue), indicating their death. Currently there isn't evidence of BLS produced by LAB active against amoebas. In this study we have shown that all the three BLS secreted by the Lactic Acid Bacteria are endowed with amoebicidal effect against Acanthamoeba polyphaga, killing the protozoan with different effectiveness and at different times of contact.